Autocrine TGF-beta signaling plays key roles in cancer progression, thereby acting as tumor suppressor in early carcinogenesis, and in stimulating invasion leading to metastasis. Most carcinomas have a deregulated response to TGF-beta, leading to inactivation of the antiproliferative response to TGF- beta. In some cases this deregulation is associated with mutations in the type I or type II TGF-beta receptors. Sumoylation, i.e. the covalent attachment of a ubiquitin-like SUMO protein, is an emerging posttranslational modification of various proteins. The role of sumoylation is often unpredictable and seems to depend on the target protein. Possible recruitment of SUMO-interacting proteins may lead to changes in the protein complex formation and functional properties of the target protein. Sumoylation is largely characterized as a modification of transcription factors and proteins involved in nuclear functions, and sumoylation of cell surface receptors has not been reported. We recently discovered that the type I TGF- beta receptor, TbRI, is sumoylated, making us hypothesize that sumoylation regulates the function of this receptor, and consequently TGF-beta signaling and the cell's response to TGF-beta. After identifying the sumoylation site in TbRI, we also found that a point mutation in TbRI, which has been found in association with metastatic breast cancer, confers a lack of sumoylation to TbRI. This leads to the hypothesis that sumoylation of the TbRI affects autocrine TGF-beta signaling and is a determinant of the invasive behavior of carcinomas. We propose three aims to define the molecular mechanism of TbRI sumoylation and its role in the cellular responses to TGF-b and the behavior of cancer cells in vivo. Aim 1 will characterize the mechanism of sumoylation of TbRI, including a search to identify the responsible E3 SUMO ligase. Aim 2 will examine the effect of TbRI sumoylation on Smad and non-Smad signaling responses and the cell's proliferation and invasion responses to TGF-b. This Aim should also define functional differences between the "metastasis- associated" TbRI mutant, wild-type TbRI and a sumoylation-deficient TbRI point mutant in the signaling and cell behavior responses to TGF-b. Finally, Aim 3 will define the effect of TbRI sumoylation and of the "metastasis-associated" TbRI point mutation on the behavior of cancer cells and on cancer progression in vivo. The proposed studies should provide a basis for the characterization of the mechanism of the TGF- beta receptor sumoylation, its role in the cell's response to TGF-beta and in cancer cell behavior and cancer progression.